核仁素转录激活子功能研究

目的：研究核仁素的转录激活子作用,探讨CD34^＋分子的基因转录调节机制。方法：分别构建核仁素的表达质粒,以及野生型和含有缺失突变的CD34^＋基因调控区质粒,启动子区下游含有荧光素酶报告基因。这些报告质粒的调控区分别是：①0．8kb野生型CD34^＋基因上游5’端序列;②缺失了127个核苷酸的CD34^＋基因上游5’端序列。应用瞬时转染试验,研究人CD34^＋基因上游5’端的转录特征,分析核仁素对CD34^＋基因的转录调节功能。结果：①含野生型CD34^＋基因上游5’端序列的报告质粒的相对荧光素酶活性约为空质粒pGL3-Basic的3倍;CD34^＋基因上游调控区的活性可被核仁素激活,其相对荧光素酶活性是空质粒的8倍。②CD34^＋基因上游5’端有127nt．缺失的报告质粒的相对荧光素酶活性与空质粒pGL3-Basic的活性相同,且与核仁素表达质粒共转染细胞时,其相对荧光素酶活性没有变化。结论：核仁素对CD34^＋基因起转录激活作用。CD34^＋基因上游5’端自-470nt．至-344nt．的区域对CD34^＋基因的表达具有重要意义。     

Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH ／ 3T3 fibroblasts

AIM: To investigate the regulatory mechanism of transforming growth factor-betaon activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS: The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-β1…     

Transcription factor binding to a putative double E-box motif represses CYP3A4 expression in human lung cells

Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5' upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (-71 to -127) that was absent in the CYP3A5 promoter. Deletion of the 57-bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the (32)P-labeled CYP3A4 57-bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double- or single-E-box binding factors (TAL1, deltaEF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5'-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5'-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.     

Organization of the CYP1A cluster on human chromosome 15: implications for gene regulation

The sequence and organization of the CYP1A cluster on human chromosome 15 was determined. A human genomic clone from a BAC library, containing both CYP1A1 and CYP1A2 genes, was isolated and sequenced. The results of Southern blot analysis using human genomic DNA were compatible with the structure of the BAC clone. The CYP1A1 and CYP1A2 genes are separated by a 23 kb segment that contains no other open reading frames. The CYP1A1 and CYP1A2 genes are in opposite orientation, revealing that the 5' flanking region is in common between the two genes. Analysis of the sequence obtained revealed the presence of xenobiotic response elements (XREs) previously reported for CYP1A1 and CYP1A2 and several additional consensus sequences for putative XREs. The presence of all the XREs upstream of both genes suggest that some of the regulatory elements known to control CYP1A1 gene expression, could also control CYP1A2 gene expression.     

Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6)

OBJECTIVES: In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2A6 gene variants are known which contribute to the highly variable expression of this enzyme among individuals. In this study we report a novel polymorphism located in the 5' flanking region (-745A > G) of the CYP2A6 gene disrupting a CCAAT box. METHODS AND RESULTS: Electrophoretic mobility shift assays (EMSA) indicated that NF-YA is part of this nuclear protein complex. Chromatin immunoprecipitation revealed that NF-Y recognizes a region of the CYP2A6 5' flanking region located between -932 and -606. EMSA showed that out of the three CCAAT boxes in the CYP2A6 promoter, with CCAAT core sequences located between -839/-835, -748/-744, and -689/-685, only the one at -748/-744 was able to compete with the nuclear protein complex binding to the -748/-744 CCAAT box. Cotransfection experiments indicated that NF-Y acts as a positive regulatory element on CYP2A6 gene regulation. EMSA demonstrated that an NF-Y consensus oligonucleotide but not the -745A > G oligonucleotide competed efficiently with binding of the protein complex to the -748/-744 CCAAT box. Promoter activity of the -745A > G variant was significantly reduced to 78% relative to the wild-type allele in HepG2 cells transfected with luciferase reporter plasmids. Finally, haplotype analysis was carried out comprising the -745A > G variant in combination with all known CYP2A6 3' and 5' flanking single nucleotide polymorphisms: -1013A > G, -48T > G, and the CYP2A6/CYP2A7 3' flank conversion. CONCLUSION: A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.     

4-Hydroxykobusin inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB and AP-1 activation

We recently isolated a novel lignan, 4-hydroxykobusin from Geranium thunbergii (Liu et al., Arch. Pharm. Res., 29, 1109-1113, 2006). Here, we studied its effect on the expression of inducible nitric oxide synthase (iNOS) gene in RAW264.7 cells. 4-Hydroxykobusin inhibited nitric oxide (NO) production in a concentration-dependent manner and blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To identify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of 4-hydroxykobusin on the transactivation of iNOS gene by luciferase reporter activity using -1.59 kb flanking region. The lignan suppressed the reporter gene activity and the LPS-induced reporter activations of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) were also significantly blocked by 4-hydroxykobusin. These findings suggest that the inhibition of LPS-induced NO formation by 4-hydroxykobusin is due to its inhibition of NF-kappaB and AP-1 activation.     

Regulation of the CYP3A4 gene by hydrocortisone and xenobiotics: role of the glucocorticoid and pregnane X receptors.

The molecular mechanisms of regulation of the CYP3A4 gene have been examined in an in vitro reporter gene system, containing -1 kb of the CYP3A4 promoter, in a HepG2 cell line. This system allows for the separate and combined transfection of expression plasmids encoding the human glucocorticoid receptor (hGR) and the human pregnane X receptor (hPXR), and, therefore, the opportunity to assess the role of these receptors in the induction process. Hydrocortisone produces a dose-dependent increase in CYP3A4 activation, a response that is increased in the presence of either receptor. Moreover, transfection of the hPXR decreased the EC(50) for hydrocortisone-dependent induction by a factor of 3.3, a response that was not changed by simultaneous cotransfection of the hGR. In addition, the hydrocortisone dose-response curve falls within the physiological blood level concentration of this steroid, implicating a regulatory role for hydrocortisone in the basal level of CYP3A4 expression. Although the responses to dexamethasone and rifampicin were both increased by both receptors, dexamethasone activation of CYP3A4 was similar for both the hGR and the hPXR, whereas rifampicin-dependent activation favored the hPXR. We conclude that regulation of the CYP3A4 gene is receptor-dependent and that hydrocortisone may function as a regulator of basal expression via the hPXR and the hGR. The implications of this latter conclusion for possible regulatory interactions between hydrocortisone and xenobiotic inducers remain to be clarified.     

Identification of novel polymorphisms within the promoter region of the human beta2 adrenergic receptor gene

By screening the 1470 bp 5' to the start codon of the human beta2 adrenergic receptor gene, we have identified a total of eight polymorphisms (-20 T-->C, -47 T-->C, -367 T-->C, -468 C-->G, -654 G-->A, -1023 G-->A, -1343 A-->G and -1429 T-->A c.f. beta2 adrenergic receptor start codon). Transient transfection of 5' flanking deletion luciferase reporter constructs demonstrated the majority of activity of the human beta2 adrenergic gene 5' flanking region to be present within a 549 bp fragment immediately upstream from the start codon. Because of linkage disequilibrium, some combinations of polymorphisms were particularly frequent. We transiently transfected COS-7 cells with luciferase constructs under the control of the 549 bp of 5' flanking DNA containing the two most frequent extended haplotypes in this region. Luciferase activity was significantly reduced in cells transfected with the 'mutant' construct (-20C, -47C, -367C, -468G) c.f. the 'wild-type' construct (-20T, -47T, -367T, -468C). These data suggest that polymorphisms have the potential to alter human beta2 adrenergic receptor gene expression.     

小鼠干扰素调节因子3启动子质粒的构建及其启动子活性分析

目的构建包含小鼠干扰素调节因子3(mIRF-3)基因启动子的质粒,并评价其启动子活性。方法以小鼠全血细胞总DNA为模板,PCR扩增mIRF-3目的片段,亚克隆此片段至pGL3-basic荧光素酶报告基因的多克隆位点,构建含mIRF-3启动子的重组报告质粒pmIRF-3。将pmIRF-3、pGL3-basic和pGL3-control质粒分别转染小鼠胚胎成纤维细胞NIH3T3后,行荧光素酶活性检测,计算相对活性单位(RLU)。生物信息学分析转录因子结合位点。结果成功构建了重组报告质粒pmIRF-3。与pGL3-basic组相比,pmIRF-3、pGL3-control组RLU明显增加(0.443±0.113vs.18.907±3.335、25.704±5.850)(P<0.01)。mIRF-3启动子区域内存在AML-1a、E2F、MZF1、CdxA、OCT-x等多个转录因子结合位点。结论 mIRF-3转录起始位点附近1479bp序列在NIH3T3细胞中具有较强的启动子活性。